SUMMARY The Family Smoking Prevention and Tobacco Control Act (FSPTCA) prohibits the addition of artificial or natural flavors to tobacco cigarettes, with the exception of menthol. While the prohibition of flavored cigarettes in 2009 set in motion a significant decline in youth tobacco use, the proportion of menthol cigarette smokers has increased, suggesting that menthol may promote initation and maintain addiction. Flavor perception results from a synthesis of stimuli carried by gustatory, olfactory and somatosensory nerves. Cooling flavors such as menthol are considered soothing,. TRPM8, a member of the TRP ion channel family, is the target of menthol and other cooling agents in cold-sensing neurons and is also activated by cold temperature. The flavor industry has developed a range of synthetic cooling agents with novel sensory properties. These synthetic cooling agents have been detected in unregulated electronic cigarette liquids and are sold by online vendors as powders or solutions to mix into E-liquids.Other flavorants may also affect properties of sensory systems, including clove flavors, flavored with eugenol, a widely used dental anesthetic, and wintergreen flavors, flavored with methyl salicylate, a topical analgesic. In our preliminary studies we show that synthetic coolants and eugenol strongly activate TRPM8 channels. We performed a chemical analysis of clove-flavored E-liquids, and revealed that some contain very high eugenol levels (>100mM). In mice we observed that menthol reduced respiratory irritation by aldehyde-flavorant containing E-liquids, suggesting that menthol facilitates inhalation. We also observed that menthol increased cotinine levels, suggesting higher nicotine intake. Based on these findings, we hypothesize that some electronic cigarette liquids contain pharmacologically active flavorants that produce vapors with counterirritant or anesthetic effects suppressing respiratory irritation and facilitating initiation of product use. The following specific aims, using analytical chemical, pharmacological and physiological methods, are designed to produce evidence for or against this hypothesis. Aim 1: Determine contents of anesthetic and cooling agents in E-cigarette liquids and condensed vapors Aim 2: Determine pharmacological effects of E-liquids and condensed vapors containing anesthetic or cooling agents on mouse and human irritant receptors and excitability of trigeminal sensory neurons Aim 3: Measure impact of vapor exposure on ventilation and the respiratory irritation response in mice. Due to the close similarities between human and rodent TRP ion channels and their flavorant pharmacology, data resulting from these studies may support efforts to regulate some flavorants as pharmacologically active agents.